1. Field of the Invention
The present invention relates to transgenic Caenorhabditis elegans overexpressing DNA methyltransferase 3a (Dnmt3a) and a method of producing the same.
2. Discussion of Related Art
DNA methylation is an epigenetic modification, and involved in the regulation of gene expression, the development of mammals, aging, and the biology of cancer. In mammals, DNA methyltransferases (DNMTs) serve to introduce methyl groups to the fifth carbon of cytosines (m5CpGs) present in cytosine-guanine (CpG) dinucleotide sequences. Such cytosine methylation serves as an important role in gene expression by providing a binding site for proteins and altering chromatin structure between DNA and histones. An epigenetic marker generated by the DNA methylation allows genes in different tissues to maintain various gene expression patterns depending on cell type.
Also, the DNA methylation is the critical factor for forming heterochromatin, involved in various processes such as the inactivation of X-chromosomes, genetic imprinting, gene silencing, and carcinogenesis, and, as another important function, stabilizes chromosomes by silencing a transcription factor so as to protect the integrity of genomes. Abnormal DNA methylation is associated with a variety of pathological events including cancer, and generally in cancer cells, characteristics such as DNA methylation, gene-specific DNA methylation, and hypermethylation of a specific promoter are observed. Particularly, it is reported that the expression of an abnormal Dnmt gene is associated with a variety of human cancers including liver cancer, prostate cancer, and breast cancer.
Also, the methylation of a regulatory DNA base sequence closely relates with gene transcriptional activity. An unmethylated CpG island is usually observed at an activated promoter site of a tissue-specific gene. In a region of a chromatin (euchromatin) at which gene transcription actively takes place, the DNA unmethylation and histone acetylation frequently occur at a promoter and the first exon site, and the methylation of histone H3 occurs at lysine residue, the fourth amino acid of histone H3. When the CpG is methylated, gene transcription may be directly influenced by interference with binding of a transcription factor to the methylated CpG, or the binding of the transcription factor may be indirectly interfered with another protein binding to the methylated CpG as a result.
In mammals, the DNMTs are divided into four types, in which the Dnmt3 family can originally methylate CpGs, but the Dnmt1 family serves to maintain a methylation pattern during the replication of DNA. The Dnmt3 family includes Dnmt3a, Dnmt3b, and Dnmt3L, in which Dnmt3a and Dnmt3b are original methyltransferases, and Dnmt3L is a modulator.
Meanwhile, the DNA methylation is phylogenetically variable. Unlike Arabidopsis thaliana which has been widely researched as a model plant, it is known that DNA methylation rarely occurs in most invertebrate animal models such as yeasts, drosophilae, and Caenorhabditis elegans (C. elegans). Particularly, genes of C. elegans are easily manipulated and have a small size, and therefore a great number of C. elegans can be simultaneously grown in vitro at a relatively low cost. C. elegans hatches from its egg and develops into an adult through four stages including L1, L2, L3, and L4, which takes only about three days, and thus it is suitable for an animal experiment. Since C. elegans has a simple body structure, consists of only 959 cells except a reproductive cell, and has a transparent body, it is easy to directly observe its inside by a microscope.
Also, the cell lineage of C. elegans from a fertilized egg to an adult has been completely identified, and as the result of the genome project, it is known that C. elegans has three times as many as the number of chromosomes of a yeast and about ⅔ of the number of chromosomes of a human. 40% of the chromosomes of C. elegans are similar to those of the human, and C. elegans shares 75% of five thousand human disease genes that have been known so far. Therefore, C. elegans is considered a good model system for human disease research.